Figure 1.
Definition of square wave pulses for eukaryotic cell transfection generated by the ELPorator 1000 device.
(A) The ELPorator 1000 device consisting of two physically independent but electrically interconnectable modules. Module 1 produces single HV square wave pulse at a maximum field strength of 5 kV/cm for 1–999 µs. Module 2 provides a single LV square wave pulse with up to 1.5 kV/cm in the range of milliseconds. Combination of HV and LV square wave pulses forms an uninterrupted combined pulse. Preferentially, a LV pulse follows a primary HV pulse. Within the ranges noted above, parameters of single or combined pulses can be defined individually. (B) Flow chart of input and output parameters within the electronic components of the individual modules.
Figure 2.
Transfection efficiency for adherent cells achieved by nucleofection compared to the ELPorator 1000 device.
For transfection of 1×106 3T3L1 cells or preadipocytes from the mesenteric fat with 2 µg pEGFP-N1, a pulse combined of a HV pulse of 3 kV/cm for 400 µs followed by a LV pulse of 1.5 kV/cm for 5 ms was applied by the ELPorator 1000 device. Alternatively, cells were transfected using the Nucleofector Kit V and program T-030 in a device for nucleofection or remained untreated. After 24 h, cell recovery and viability were assessed by trypan blue exclusion. Transfection efficiency was determined by flow cytometry as percentage of EGFP+ cells. Mean values ± SEM; n = 5.
Figure 3.
Impact of pulse composition and plasmid DNA amount on the transfection efficiency for primary murine preadipocytes.
(A) 3T3L1 cells were transfected with 2 µg pEGFP-N1 using the ELPorator 1000 device for single HV or LV pulses or for a combined HV/LV pulse (HV: 3 kV/cm, 400 µs; LV: 1.5 kV/cm, 5 ms). Efficiency was estimated by flow cytometry from the percentages of EGFP+ cells and from individual EGFP expression levels after 24 h. Mean values ± SEM, n = 2. (B) Primary murine preadipocytes were transfected with different amounts of pEGFP-N1 vector using a single HV pulse (3 kV/cm, 400 µs; white circles) or a combined HV/LV pulse (3 kV/cm, 400 µs and 1.5 kV/cm, 5 ms; black circles). Transfection efficiency was determined from the percentages of EGFP+ cells and EGFP expression levels after 24 h. Mean values ± SEM; n = 3.
Table 1.
Transfection efficiencies and viable cell recovery in suspension cell lines.
Figure 4.
Transfection efficiency of defined lymphocytic and monocytic cell subpopulations within mixed human peripheral blood mononuclear cells.
Freshly prepared human PBMC (3×106) were transfected with 4 µg pEGFP-N1. A single HV pulse of 4.5 kV/cm for 400 µs or combined with a LV pulse of 0.75 kV/cm for 3 ms was applied by the ELPorator 1000 device. Non-treated PBMC served as controls. (A) Cells were incubated with directly labeled monoclonal antibodies and overall distribution of the subpopulations within mixed PBMC preparations, percentages of EGFP+ cells, and EGFP expression levels within the individual populations were determined by multi-color flow cytometry after 24 h. Mean values from triplicate determinations; n = 6. (B) Viability of treated cells was assessed after 24 h by propidium iodide staining. Mean values from triplicate determinations; n = 6.
Figure 5.
Long-term outcome of transfection of primary preadipocytes with GAPDH-specific siRNA.
Primary preadipocytes were transfected with 120 pmol Cy3-labeled GAPDH-specific siRNA or a non-coding siRNA for control using a single HV pulse (3 kV/cm, 400 µs) or a combined HV/LV pulse (3 kV/cm, 400 µs and 1.5 kV/cm, 5 ms) produced by the ELPorator 1000 device. (A) After 24, 48 and 72 h, copy numbers of GAPDH-specific mRNA were determined by quantitative PCR in comparison to a cloned plasmid standard. Mean values ± SEM; n = 6. (B) As directly assessed by flow cytometry 5 h after transfection, uptake of Cy3-labeled GAPDH-specific siRNA by preadipocytes was determined via the MFI. Representative histogram and mean values ± SEM; n = 4.
Figure 6.
Functionality of siRNA in transfected cells.
Primary preadipocytes were transfected with 100 pmol TLR4-specific siRNA or non-coding control siRNA using the combined HV/LV pulse strategy (3 kV/cm, 400 µs; 1.5 kV/cm, 5 ms). One day after transfection, cultures received 1 µg/ml LPS or 200 nM LT-DAP for additional 24 h before IL-6 production was determined from the cell culture supernatants. Bars represent the mean of n = 3 ± SEM.